Electromembrane Extraction in Suspect Screening of Polar Organic Xenobiotics and their Metabolites in Human Urine: A New Approach to Enhance Compound Annotation?

Suspect and nontarget screening (SNTS) methodologies using human urine are invaluable strategies for understanding the human exposome. However, very polar organic compounds are often overlooked in those methods due to challenges in sample preparation and chromatographic analysis. Although "dilute-and-shoot" (DS) followed by mixed-mode liquid chromatography-high-resolution mass spectrometry (MMLC-HRMS) might be deemed suitable, complementary strategies are needed to enhance SNTS and expand compound identification. In this context, the potential of nonsupported microelectromembrane extraction (μ-EME) is thoroughly studied as a supplement to DS-MMLC-HRMS. It was demonstrated that μ-EME-MMLC-HRMS enables the refinement of suspect screening results from a 24 h pooled human urine sample. The selective extraction capability of μ-EME for charged analytes, compared to DS, allowed the identification of 24 false positives and 4 false negatives. The confidence level of 6 suspects was also enhanced through μ-EME interpretation. Moreover, nine suspects were identified exclusively in μ-EME experiments due to the urine cleanup provided by that technique. Notably, suspects containing carboxylic acid groups (phase II metabolites) and amines were particularly well-annotated by μ-EME employing selective extraction conditions for acids and bases, respectively. Thus, μ-EME proves to be a confirmatory dimension in MMLC-based SNTS approaches.